The effects of Ca++ concentration and degree of filament overlap on the kinetics of myosin cross-bridge interaction with actin will be studied in single fibers of chemically skinned rabbit psoas, and results will be correlated with those form biochemical studies. Intact fibers of frog semitendinosus and crayfish walking leg muscles are optionally employed for comparison. We aks whether Ca++ affects kinetic parameters in a graded manner, or whether it acts as a "switch", hence simply recruiting more cross-bridges into the active pool. We also ask whether muscle compliance is localized in the cross-bridge or in structures in series with it. We attempt to explain the complex stiffness and rate constant data with a scheme consistent also with the results of biochemical studies. In this way we can evaluate the applicability of information gained from reconstituted biochemical systems (no load) to structured skinned muscle systems (with load). Our proposed studies also cover (i) myosin isoenzyme distdribution, (ii) low MgATP concentration, (iii) low ionic strength, (iv) ATP buffering capacity, (v) sarcomere length and filament overlap, (vi) reagent that "shrink" the myofilament lattice, and (vii) model development. We currently employ the sinusoidal analysis technique, but we will start undertaking the step length change technique in the 2nd year and the force-velocity experiments in the 4th year. We will also improve the frequency response of our experimental system and search for new transfer function(s) to describe the complex stiffness data.